Universal anti-tag chimeric antigen receptor-expressing T cells and methods of treating cancer

ABSTRACT

The present invention provides a universal, yet adaptable, anti-tag chimeric antigen receptor (AT-CAR) system which provides T cells with the ability and specificity to recognize and kill target cells, such as tumor cells, that have been marked by tagged antibodies. As an example, αFITC-CAR-expressing T cells have been developed that specifically recognize various human cancer cells when those cells are bound by cancer-reactive FITC-labeled antibodies. The activation of αFITC-CAR-expressing T cells is shown to induce efficient target lysis, T cell proliferation, and cytokine/chemokine production. The system can be used to treating subjects having cancer.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant NumbersCA140917 and HL088954 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The invention relates to T cell-based anti-cancer therapeutics andmethods of using the therapeutics in the treatment of cancer.

BACKGROUND OF INVENTION

The ability to make a universal yet versatile system to generate T cellsthat are capable of recognizing various types of cancers has importantclinical implications for the use of T cell-based therapies. One currentstrategy incorporates the use of genetic engineering to express achimeric antigen receptor (CAR) on T cells. The extracellular domain ofa typical CAR consists of the V_(H) and V_(L) domains—single-chainfragment variable (scFv)—from the antigen binding sites of a monoclonalantibody. The scFv is linked to a flexible transmembrane domain followedby a tyrosine-based activation motif such as that from CD3ζ (Sadelain etal. Curr. Opin. Immunol. 21, 215-223 (2009); Gross et al. Proc. Natl.Acad. Sci. USA 86, 10024-10028 (1989); Ertl et al. Cancer Res. 71,3175-3181 (2011). The so-called second and third generation CARs includeadditional activation domains from co-stimulatory molecules such as CD28and CD137 (41BB) which serve to enhance T cell survival andproliferation. CAR T cells offer the opportunity to seek out and destroycancer cells by recognizing tumor-associated antigens (TAA) expressed ontheir surface (Sadelain et al. Curr. Opin. Immunol. 21, 215-223 (2009)).As such, the recognition of a tumor cells occurs via an MHC-independentmechanism. Various preclinical and early-phase clinical trials highlightthe efficacy of CAR T cells to treat cancer patients with solid tumorsand hematopoietic malignancies (Kershaw et al. Clin. Cancer Res. 12,6106-6115 (2006); Lamers et al. J. Clin. Oncol. 24, e20-e22 (2006);Morgan et al. Mol. Ther. 18, 843-851 (2010); Pule et al. Nat. Med. 14,1264-1270 (2008); Till et al. Blood 112, 2261-2271 (2008)).

Despite of the promise that CAR T cells might have in treating cancerpatients there are several limitations to the generalized clinicalapplication of CAR T cells. First, since no single tumor antigen isuniversally expressed by all cancer types, scFv in CAR needs to beconstructed for each tumor antigen to be targeted. Second, the financialcost and labor-intensive tasks associated with identifying andengineering scFvs against a variety of tumor antigens poses a majorchallenge. Third, tumor antigens targeted by CAR could be down-regulatedor mutated in response to treatment resulting in tumor evasion. Sincecurrent CAR T cells recognize only one target antigen, such changes intumors negate the therapeutic effects. Therefore, the generation of CARTcells capable of recognizing multiple tumor antigens is highly desired.Finally, CAR T cells react with target antigen weakly expressed onnon-tumor cells, potentially causing severe adverse effects (Morgan etal. Mol. Ther. 18, 843-851 (2010)). To avoid such “on-target off-tumor”reaction, use of scFvs with higher specificity to tumor antigen isrequired. And although ongoing studies are focused on generating methodsto shut off CAR T cells in vivo this system has yet to be developed andmight pose additional inherent challenges.

Modifications to existing CAR T cell systems that address and overcomethe hurdles currently preventing development of the systems intoeffective means of in vivo treatment are therefore needed.

BRIEF SUMMARY OF INVENTION

The present invention provides a universal, yet adaptable, anti-tagchimeric antigen receptor (AT-CAR)-expressing T cell system that fullyaddresses the deficiencies of current systems. The system uses a genetherapy platform to generate immune cells capable of recognizing variouscancers types and that have broad and valuable clinical implications forthe use of T cell-based therapies. As disclosed herein, a versatileAT-CAR system which grants T cells specificity to recognize and bindtagged proteins, such as antibodies, has been developed.

For example, and as further described herein, αFITC-CAR-expressing humanT cells have been developed that specifically recognize various humancancer cells when those cells are bound by cancer-reactive FITC-labeledantibodies. The activation of αFITC-CAR-expressing T cells is shown toinduce efficient target lysis, T cell proliferation, andcytokine/chemokine production in vitro and ex vivo. In vivo,αFITC-CAR-expressing T cells plus FITC-cetuximab (Ctx) are shown todelay colon cancer tumor establishment but lead to the selection oftumor-associated antigen (TAA)-negative cancer cells. Using a pancreatictumor model with uniform TAA expression, αFITC-CAR-expressing T cellswere observed to eradicate an established tumor and prevent tumorgrowth. This ‘off-the-shelf’ system advances existing CAR technologythrough its potential to target various tagged proteins in the treatmentof cancer patients.

In certain embodiments, the invention is drawn to a method of treatingcancer in a subject, comprising: (a) administering a formulation oftagged proteins to a subject in need of treatment, wherein the taggedproteins bind a cancer cell in the subject, and (b) administering atherapeutically-effective population of anti-tag chimeric antigenreceptor (AT-CAR)-expressing T cells to the subject, wherein theAT-CAR-expressing T cells bind the tagged proteins and induce cancercell death, thereby treating cancer in a subject.

In a related embodiment, the invention is drawn to a method of treatingcancer in a subject, comprising: (a) administering one or moreformulations of tagged proteins to a subject in need of treatment,wherein the tagged proteins bind a cancer cell in the subject, and (b)administering one or more therapeutically-effective populations ofAT-CAR-expressing T cells to the subject, wherein the AT-CAR-expressingT cells bind the tagged proteins and induce cancer cell death, therebytreating cancer in a subject.

In a further related embodiment, the invention is drawn to a method oftreating cancer in a subject, comprising: (a) administering at least twoformulations of tagged proteins to a subject in need of treatment,wherein the tagged proteins bind a cancer cell in the subject, and (b)administering at least two therapeutically-effective populations ofAT-CAR-expressing T cells to the subject, wherein the AT-CAR-expressingT cells bind the tagged proteins and induce cancer cell death, therebytreating cancer in a subject.

In particular aspects of the embodiments of the invention, the tag ofeach formulation of tagged proteins is the same or different and the tagis selected from the group consisting of fluorescein isothiocyanate(FITC), streptavidin, biotin, histidine, dinitrophenol, peridininchlorophyll protein complex, green fluorescent protein, phycoerythrin(PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalaninephosphatase, glucose oxidase, and maltose binding protein.

In particular aspects of the embodiments of the invention, the proteinof each formulation of tagged proteins is the same or different and theprotein is an antibody or an antigen-binding fragment thereof. Inpreferred aspects, the antibody or antigen-binding fragment thereof iscetuximab, nimotuzumab, panitumumab, retuximab, omalizumab, tositumomab,trastuzumab, gemtuzumab, alemtuzumab, bevacuzimab or an antigen-bindingfragment of any one thereof.

In particular aspects of the embodiments of the invention, the AT-CAR ofeach population of AT-CAR-expressing T cells is the same or differentand the AT-CAR comprises a tag-binding domain, a transmembrane domain,and an activation domain. In preferred aspects, the tag-binding domainis an antibody or an antigen-binding fragment thereof. In preferredaspects, the tag-binding domain specifically binds FITC, biotin, PE,histidine or streptavidin. In preferred aspects where the tag-bindingdomain is antigen-binding fragment, the antigen-binding fragment is asingle chain variable fragment (scFv), such as a scFv that specificallybinds FITC, biotin, PE, histidine or streptavidin. In preferred aspectsthe transmembrane domain is the hinge and transmembrane regions of thehuman CD8α chain. In preferred aspects, the activation domain comprisesone or more of the cytoplasmic region of CD28, the cytoplasmic region ofCD137 (41BB), OX40, HVEM, CD3ζ and FcRε.

In particular aspects of the embodiments of the invention, the T cellsof each population of AT-CAR-expressing T cells are the same ordifferent and wherein the T cells are selected from the group consistingof T cells of any HLA-background from peripheral blood mononuclear cells(PBMC), T cells isolated from a tumor explant of the subject, andintratumoral T cells of the subject.

In particular aspects of the embodiments of the invention, the T cellsof each population of AT-CAR-expressing T cells consist of HLA-A2+peripheral blood mononuclear cells (PBMC).

In particular aspects of the embodiments of the invention, theformulation(s) of tagged proteins are administered to the subject priorto administration of the therapeutically-effective population(s) ofAT-CAR-expressing T cells.

In particular aspects of the embodiments of the invention, theformulation(s) of tagged proteins are administered to the subjectconcurrently with administration of the therapeutically-effectivepopulation(s) of AT-CAR-expressing T cells.

In particular aspects of the embodiments of the invention, theformulation(s) of tagged proteins are administered to the subject afteradministration of the therapeutically-effective population(s) ofAT-CAR-expressing T cells.

In particular aspects of the embodiments of the invention, theformulation(s) of tagged proteins and the therapeutically-effectivepopulation(s) of AT-CAR-expressing T cells are administered to thesubject in any order.

In particular aspects of the embodiments of the invention,AT-CAR-expressing T cell binding to the tagged proteins inducescytolytic activation of the T cells.

In particular aspects of the embodiments of the invention, the subjectis a human.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedherein, which form the subject of the claims of the invention. It shouldbe appreciated by those skilled in the art that any conception andspecific embodiment disclosed herein may be readily utilized as a basisfor modifying or designing other formulations for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent formulations do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thatany description, figure, example, etc. is provided for the purpose ofillustration and description only and is by no means intended to definethe limits the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Anti-FITC-CAR expression, characterization and in vitrofunctionality. (A) Diagram of the anti-FITC-CAR (αFITC-CAR). Boxeddiagram on left shows the genetic elements included in thepolynucleotide engineered to express αFITC-CAR, beginning with a 5′-longterminal repeat, and followed by the coding region for anti-FITC scFv,CD28 transmembrane domain, CD28, 41BB, zeta chain (ζ) and a 3′-longterminal repeat. Figure on right shows the αFITC-CAR polypeptidetraversing the T cell membrane and position to bind a tumor-reactiveantibody labeled with FITC. (B) HLA-A2⁺ PBMCs were activated withanti-CD3 antibodies in the presence of IL-2 followed by transductionwith αFITC-CAR retrovirus. αFITC-CAR expression was determined bystaining cells with CD3 and FITC-conjugated cetuximab (FITC-Ctx) orFITC-conjugated dextran beads (FITC-Dex). Cetuximab is an antibody withbinding specificity for EGFR-expressing tumor cells. (C) αFITC-CARfunctionality on transduced and control T cells was examined inproliferation assays using plate-bound FITC-Ctx, FITC-Dex or Ctx. (D)The proliferation of αFITC-CAR T cells and control T cells was measuredin response to stimulation with SW480 colon cancer cells stained withtitrating concentrations of FITC-Ctx. (E) T cell cytotoxicity byαFITC-CAR and control T cells was measured against SW480 colon cancercells stained with FITC-Ctx or FITC-mouse IgG. (F) αFITC-CARcytotoxicity was measured against Panc 6.03 stained with unlabeled Ctxand Her2 antibodies or stained with FITC-Ctx or FITC-Her2 at variouseffector to target ratios. Alternatively AU565 breast cancer cells werestained with FITC-Her2 or unlabeled Her2 mAb. All data arerepresentative of three or more independent experiments each yieldingidentical trends.

FIG. 2. αFITC-CAR T cells delay tumor establishment but promote thegrowth of antigen-negative tumor cells. (A) SW480 human colon cancercells were injected subcutaneously into NSG mice followed by injectionwith FITC-Ctx (i.p.) one day later. Twenty four hours after, 5×10⁶αFITC-CAR T cells were injected into the tail vein. FITC-Ctx wasinjected (i.p.) weekly for three weeks. Data are representative of twoor three experiments (three or more mice per group). *, P≤0.02, ANOVA;n.s. not significant. (B) The percentage of CD3⁺ αFITC-CAR positive andnegative T cells was determined by flow cytometry. The averagepercentage of αFITC-CAR⁺ T cells prior to injection or from the tumorexplant of four mice is shown. (C) Tumor explants were finely minced andcultured in trypsin for 2 hours at 37° C. followed by T cell enrichmentusing a negative selection kit. T cells were co-cultured with SW480colon cancer cells which had been pulsed with FITC-Ctx or Ctx (0.5μg/1×10⁶ cells). Three days later proliferation was determined by[³H]thymidine uptake (±SD). (D) Alternatively, cytokine and chemokineproduction was measured using a Milliplex array 72 hours afterstimulation. (E) EGFR expression on SW480 tumor explants or SW480 cellstaken from tissue culture was examined by flow cytometry using FITC-Ctx.

FIG. 3. αFITC-CAR T cells prevent tumor growth and eradicate establishedtumors. (A) EGFR expression on the pancreatic human cancer cells (Panc6.03) was determined by staining cells with FITC-Ctx or with controlantibody FITC-mIgG and analyzed by flow cytometry. (B) In theprophylactic tumor model, Panc 6.03 cells were injected subcutaneouslyinto NSG mice (n=5) followed by injection with FITC-Ctx (i.p.) one daylater. Twenty four hours later 5×10⁶ αFITC-CAR T cells were injectedinto the tail vein. 25 μg of FITC-Ctx or Ctx was injected (i.p.) weeklyfor three weeks. Tumor growth and mouse survival is shown. (C) In thetherapeutic model Panc 6.03 tumors were grown to sizes between 3-10 mm²and then injected (i.p.) with 25 μg of FITC-Ctx or Ctx every week forthree weeks. One day after the first injection of FITC-Ctx, mice wereadministered 5×10⁶ αFITC-CAR T cells via tail vein injection. *, P≤0.02,ANOVA; n.s. not significant.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found, for example, in Benjamin Lewin, Genes VII, published by OxfordUniversity Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); TheEncyclopedia of Molecular Biology, published by Blackwell Publishers,1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by Wiley,John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technicalreferences.

As used herein, “a” or “an” may mean one or more. As used herein whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more. Furthermore, unless otherwise required bycontext, singular terms include pluralities and plural terms include thesingular.

As used herein, “about” refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited value) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). In some instances, the term“about” may include numerical values that are rounded to the nearestsignificant figure.

II. The Present Invention

An adaptable generation of CARs has been developed which allows for thewidespread use of personalized T cell-based immunotherapy. Human T cellshave been engineered to express an anti-FITC CAR-CD28-41BB-CD3ζ(referred to αFITC-CAR). This platform takes advantage of the highaffinity interaction between the anti-FITC scFv (on the cell's surface)and FITC as well as the ability to cheaply and easily conjugated FITCmolecules (or other tags) to any anti-cancer-based monoclonal antibodysuch as cetuximab (anti-EGFR), retuximab (anti-CD20) and herceptin(anti-Her2) used to treat patients with various types of cancers. Thissystem allows for extreme specificity to the antigen and is accompaniedby robust effector function and proliferative capacity with nocross-reactivity to ‘self-antigens’.

Effector cells. The effector cells used in the methods of the presentinvention may be autologous, syngeneic or allogeneic, with the selectiondependent on the disease to be treated and the means available to do so.Suitable populations of effector cells that may be used in the methodsinclude any immune cells with cytolytic activity, such as T cells.Exemplary sub-populations of T cells include, but are not limited tothose expressing CD3⁺ including CD3⁺CD8⁺ T cells, CD3⁺CD4+ T cells, andNKT cells. In one aspect, the T cells are HLA-A2+ peripheral bloodmononuclear cells (PBMC) but the T cells can be of any HLA backgroundfrom PBMCs and utilized in an autologous, syngeneic or allogeneicsystem. T cells may also be isolated from any source, including from atumor explant of the subject being treated or intratumoral T cells ofthe subject being treated. For the sake of convenience, the effectorcells are commonly referred to herein as T cells, but it should beunderstood that any reference to T cells, unless otherwise indicated, isa reference to all effector cell types as defined herein.

Anti-tag chimeric antigen receptor (AT-CAR). The skilled artisan willappreciate that the anti-tag chimeric antigen receptor (AT-CAR)expressed by the T cells used in the methods of the present inventionallow for great flexibility. The sole requirements for the AT-CARs usedin the methods are (i) that the AT-CAR has binding specificity for aparticular tag that can be conjugated to a protein (such as an antibody)that binds to a tumor-associated antigen (TAA), and (ii) that T cellscan be engineered to express the AT-CAR. Additional features that arepreferred, but not required, include (i) that the AT-CAR includes anactivation domain that induces efficient target lysis upon T cellbinding and activation, and (ii) the ability to replace the scFv portionof the AT-CAR with one of specificity to other tags, such as biotin orphycoerythrin, which can be conjugated to target (i.e., tumor)-reactiveproteins such as antibodies to be used in vivo.

In particular aspects, the AT-CAR comprises three domains. The firstdomain is the tag-binding domain. This domain is typically present atthe amino terminal end of the polypeptide that comprises the AT-CAR.Locating the tag-binding domain at the amino terminus permits thetag-binding domain unfettered access to the tagged protein that is boundto the target cell. As used herein, the tag-binding domain is typicallyan antibody or an antigen-binding fragment thereof. The identity of theantibody or fragment is only limited by the identity of the tag of thetagged protein. For example, the antibodies may be obtained from anyspecies of animal, though preferably from a mammal such as a human,simian, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig,dog or cat. Preferably the antibodies are human or humanized antibodies.Nor is there a limitation on the particular class of antibody that maybe used, including IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgEantibodies. Antibody fragments include single-chain variable fragment(scFv), single chain antibodies, F(ab′)₂ fragments, Fab fragments, andfragments produced by an Fab expression library, with the onlylimitation being that the antibody fragments retain the ability to bindthe selected tag.

The antibodies may also be polyclonal, monoclonal, or chimericantibodies, such as where an antigen binding region (e.g., F(ab′)2 orhypervariable region) of a non-human antibody is transferred into theframework of a human antibody by recombinant DNA techniques to produce asubstantially human molecule. Antigen-binding fragments, such as scFv,may be prepared therefrom.

One advantage of the AT-CARs of the present invention is that they canbe produced using commercially-available antibodies. Alternatively,antibodies to a selected tag may be produced. For the production ofantibodies, various hosts including, but not limited to, goats, rabbits,rats, mice, humans, etc., can be immunized by injection with aparticular protein or any portion, fragment, or oligopeptide thatretains immunogenic properties of the protein. Depending on the hostspecies, various adjuvants can be used to increase the immunologicalresponse. Such adjuvants include, but are not limited to, detoxifiedheat labile toxin from E. coli, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacteriumparvum are also potentially useful adjuvants.

Antibodies and fragments thereof can be prepared using any techniquethat provides for the production of antibody molecules, such as bycontinuous cell lines in culture for monoclonal antibody production.Such techniques include, but are not limited to, the hybridoma techniqueoriginally described by Koehler and Milstein (Nature 256:495-497(1975)), the human B-cell hybridoma technique (Kosbor et al., ImmunolToday 4:72 (1983); Cote et al., Proc Natl. Acad. Sci 80:2026-2030(1983)), and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96(1985)).

Techniques developed for the production of “chimeric antibodies,” i.e.,the splicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activity,can also be used (Morrison et al., Proc Natl. Acad. Sci 81:6851-6855(1984); Neuberger et al., Nature 312:604-608(1984); Takeda et al.,Nature 314:452-454(1985)). Alternatively, techniques described for theproduction of single chain antibodies, such as disclosed in U.S. Pat.No. 4,946,778, incorporated herein by reference in its entirety, can beadapted to produce tag-specific single chain antibodies.

In one aspect, the tag-binding domain is a single-chain variablefragment (scFv). A scFv comprises the variable regions of the heavy (VH)and light chains (VL) of an antibody, typically linked via a shortpeptide of ten to about 25 amino acids. The linker can either connectthe N-terminus of the VH with the C-terminus of the VL, or vice versa.

As indicated above, the binding specificity of the tag-binding domainwill depend on the identity of the tag that is conjugated to the proteinthat is used to bind target cells. For example, when the tag is FITC(Fluorescein isothiocyanate), the tag-binding domain may constitute ananti-FITC scFv. Alternatively, when the tag is biotin or PE(phycoerythrin), the tag-binding domain may constitute an anti-biotinscFv or an anti-PE scFv.

The second domain is a transmembrane (TM) domain. The TM domain allowsthe CAR to be anchored into the cell membrane of the T cell. ExemplaryTM domains include, but are not limited to, the hinge and transmembraneregions of the human CD8α chain.

The third domain, when present, is the T cell activation domain. Thisdomain aids in T cell activation upon binding of the CAR to the taggedprotein that is bound to the target cell. T cell activation includesinduction of cytokine and chemokine production, as well as activation ofthe cytolytic activity of the cells. Exemplary T cell activation domainsinclude, but are not limited to, the cytoplasmic regions of CD28, CD137(41BB), OX40 and HVEM which serve to enhance T cell survival andproliferation; and CD3ζ and FcRε which induce T cell activation. One ormore than one T cell activation domain may be included in the CAR, suchas two, three, four or more T cell activation domains.

AT-CAR T cell production. T cells may be engineered to express AT-CARsby means readily known to the skilled artisan. Generally, apolynucleotide vector is constructed that encodes the AT-CAR and thevector is transfected into a population of T cells. The cells are thengrown under conditions promoting expression of the AT-CAR by the Tcells. Successful transfection (or transduction which refers toviral-mediated gene integration) and display of AT-CARs by T cells isconducted via conventional means, some of which are disclosed in theExamples herein.

In one aspect, T cells may be engineered to produce AT-CARs by firstconstructing a retroviral vector encoding a selected AT-CAR. Anexemplary retroviral vector includes, but is not limited to, the vectorbackbone pMSGV1-CD8-28BBZ, which is derived from pMSGV (murine stem cellvirus-based splice-gag vector). DNA sequencing can be used to confirmproper construction of the vector before transfection of T cells.Retroviral transduction may be performed using known techniques, such asthat of Johnson et al. (Blood 114, 535-546 (2009)). The surfaceexpression of AT-CAR on transduced T cells may be determined, forexample, by flow cytometry after staining cells with tag-conjugatedprotein or tag-conjugated dextran beads. The tag portion of the proteinor beads will be bound by the tag-binding domain of the CAR expressed bythe cells.

AT-CART cell administration. Populations of AT-CAR-expressing T cellsmay be formulated for administered to a subject using techniques knownto the skilled artisan. Formulations comprising populations ofAT-CAR-expressing T cells may include pharmaceutically acceptableexcipient(s). Excipients included in the formulations will havedifferent purposes depending, for example, on the nature of thetag-binding domain comprising the AT-CAR, the subpopulation of T cellsused, and the mode of administration. Examples of generally usedexcipients include, without limitation: saline, buffered saline,dextrose, water-for-infection, glycerol, ethanol, and combinationsthereof, stabilizing agents, solubilizing agents and surfactants,buffers and preservatives, tonicity agents, bulking agents, andlubricating agents. The formulations comprising populations ofAT-CAR-expressing T cells will typically have been prepared and culturedin the absence of any non-human components, such as animal serum (e.g.,bovine serum albumin).

A formulation may include one population of AT-CAR-expressing T cells,or more than one, such as two, three, four, five, six or morepopulations of AT-CAR-expressing T cells. The different populations ofAT-CAR T cells can vary based on the identity of the tag-binding domain,the identity of the activation domain, the identity of the subpopulationof T cells, or a combination thereof. For example, a formulation maycomprise populations of AT-CAR-expressing T cells that recognize andbind to one, or more than one, such as two, three, four, five, six ormore different tagged proteins. As an example, a formulation maycomprise populations of AT-CAR-expressing T cells that recognize andbind FITC, biotin and PE. Thus, in this example, the formulationcomprises three different populations of AT-CAR-expressing T cells thatrecognize and bind cells tagged by FITC-conjugated antibodies,biotin-conjugated antibodies, and PE-conjugated antibodies. Thisformulation would therefore comprise αFITC-CAR-expressing T cells,abiotin-CAR-expressing T cells and αPE-CAR-expressing T cells.

The formulations comprising population(s) of AT-CAR T cells may beadministered to a subject using modes and techniques known to theskilled artisan. Exemplary modes include, but are not limited to,intravenous injection. Other modes include, without limitation,intratumoral, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo),intramuscular (i.m.), intraperitoneal (i.p.), intra-arterial,intramedulary, intracardiac, intra-articular (joint), intrasynovial(joint fluid area), intracranial, intraspinal, and intrathecal (spinalfluids). Any known device useful for parenteral injection or infusion ofthe formulations can be used to effect such administration.

The formulations comprising population(s) of AT-CAR-expressing T cellsthat are administered to a subject comprise a number ofAT-CAR-expressing T cells that is effective for the treatment and/orprophylaxis of the specific indication or disease. Thus,therapeutically-effective populations of AT-CAR-expressing T cells areadministered to subjects when the methods of the present invention arepracticed. In general, formulations are administered that comprisebetween about 1×10⁴ and about 1×10¹⁰ AT-CAR-expressing T cells. In mostcases, the formulation will comprise between about 1×10⁵ and about 1×10⁹AT-CAR-expressing T cells, from about 5×10⁵ to about 5×10⁸AT-CAR-expressing T cells, or from about 1×10⁶ to about 1×10⁷AT-CAR-expressing T cells. However, the number of AT-CAR-expressing Tcells administered to a subject will vary between wide limits, dependingupon the location, source, identity, extent and severity of the cancer,the age and condition of the individual to be treated, etc. A physicianwill ultimately determine appropriate dosages to be used.

Tagged proteins. Tagged proteins are administered to a subject prior to,or concurrent with, or after administration of the AT-CAR-expressing Tcells. The tagged proteins bind to target cells in the subject. Ingeneral, the “protein” portion of the tagged protein is the portion ofthe molecule that binds to the target cell. For example, the protein maybe an antibody that binds to a tumor-associated antigen (TAA) or a tumorspecific antigen (TSA) expressed by the target cell. However, the“protein” may be any molecule that binds to a target cell. Exemplaryproteins include, but are not limited to, anti-cancer-based monoclonalantibodies such as cetuximab (anti-EGFR), nimotuzumab (anti-EGFR),panitumumab (anti-EGFR), retuximab (anti-CD20), omalizumab (anti-CD20),tositumomab (anti-CD20), trastuzumab (anti-Her2), gemtuzumab(anti-CD33), alemtuzumab (anti-CD52), and bevacuzimab (anti-VEGF).

The “tag” portion of the tagged protein is only constrained by being amolecular that can be recognized and specifically bound by the AT-CAR,specifically, the tag-binding domain of the AT-CAR. Exemplary tagsinclude, but are not limited to, fluorescein isothiocyanate (FITC),dinitrophenol, peridinin chlorophyll protein complex, green fluorescentprotein, biotin, phycoerythrin (PE), histidine, streptavidin, horseradish peroxidase, palmitoylation, nitrosylation, alkalaninephosphatase, glucose oxidase, Glutathione S-transferase, maltose bindingprotein, and any types of fluorescent materials including quantum dotnanocrystals.

Thus, in some aspects, the tagged proteins include FITC-conjugatedantibodies, biotin-conjugated antibodies, PE-conjugated antibodies,histidine-conjugated antibodies and streptavidin-conjugated antibodies,where the antibody binds to a TAA or a TSA expressed by the targetcells. For example, the tagged proteins of the present inventioninclude, but are not limited to, FITC-conjugated cetuximab,FITC-conjugated retuximab, FITC-conjugated herceptin, biotin-conjugatedcetuximab, biotin-conjugated retuximab, biotin-conjugated herceptin,PE-conjugated cetuximab, PE-conjugated retuximab, PE-conjugatedherceptin, histidine-conjugated cetuximab, histidine-conjugatedretuximab, histidine-conjugated herceptin, streptavidin-conjugatedcetuximab, streptavidin-conjugated retuximab, andstreptavidin-conjugated herceptin. Alternatively, the AT-CAR cells canbe redirected to target and/or destroy vascular cells feeding the tumor.For example, T cells expressing αFITC-VEGF as the AT-CAR can targetendothelial vascular cells to which FITC-tagged VEGF is bound, where theFITC-tagged VEGF is bound by the VEGF receptor.

In some aspects, a protein lacking a tag may be used as the taggedprotein. For example, a naked (tagless) protein (such as an antibody)that binds to a TAA or a TSA on a target cell may be used as the taggedprotein. Under such circumstances, the AT-CAR will recognize andspecifically bind the protein. As an example, the tag-binding domain maybe an antibody or antigen-binding fragment thereof that recognizes andbinds a second antibody, where the second antibody functions as thetagged protein and where the second antibody lacks a tag.

The tag may be conjugated to the proteins using techniques such aschemical coupling and chemical cross-linkers. Alternatively,polynucleotide vectors can be prepared that encode the tagged proteinsas fusion proteins. Cell lines can then be engineered to express thetagged proteins, and the tagged proteins can be isolated from culturemedia, purified and used in the methods disclosed herein.

The tagged proteins may be formulated for administered to a subjectusing techniques known to the skilled artisan. Formulations of thetagged proteins may include pharmaceutically acceptable excipient(s).Excipients included in the formulations will have different purposesdepending, for example, on the nature of the tag, the protein, and themode of administration. Examples of generally used excipients include,without limitation: saline, buffered saline, dextrose,water-for-infection, glycerol, ethanol, and combinations thereof,stabilizing agents, solubilizing agents and surfactants, buffers andpreservatives, tonicity agents, bulking agents, and lubricating agents.

A formulation of tagged proteins may include one type of tagged protein,or more than one, such as two, three, four, five, six or more types oftagged proteins. The different types of tagged proteins can vary basedon the identity of the tag, the identity of the protein, or both. Forexample, a formulation comprising three types of tagged protein mightinclude FITC-conjugated cetuximab, FITC-conjugated rituximab andFITC-conjugated herceptin, or FITC-conjugated cetuximab,biotin-conjugated cetuximab, and PE-conjugated cetuximab.

The tagged proteins may be administered to a subject using modes andtechniques known to the skilled artisan. Exemplary modes include, butare not limited to, intravenous, intraperitoneal, and intratumoralinjection. Other modes include, without limitation, intradermal,subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.),intra-arterial, intramedulary, intracardiac, intra-articular (joint),intrasynovial (joint fluid area), intracranial, intraspinal, andintrathecal (spinal fluids). Any known device useful for parenteralinjection or infusion of the formulations can be used to effect suchadministration.

Formulations comprising the tagged proteins are administered to asubject in an amount which is effective for treating and/or prophylaxisof the specific indication or disease. In general, formulationscomprising at least about 0.1 mg/kg to about 100 mg/kg body weight ofthe tagged proteins are administered to a subject in need of treatment.In most cases, the dosage is from about 1 mg/kg to about 10 mg/kg bodyweight of the tagged proteins daily, taking into account the routes ofadministration, symptoms, etc. As an example, tagged-bevacizumab isadministered in a dosage of from about 2.5 to about 5 mg/kg. As afurther example, tagged-cetuximab is administered in a dosage rangingfrom about 100 to about 400 mg/m². However, the amount of tagged proteinin formulations administered to a subject will vary between wide limits,depending upon the location, source, identity, extent and severity ofthe cancer, the age and condition of the individual to be treated, etc.A physician will ultimately determine appropriate dosages to be used.

Cancer. The present invention relates to methods of treating a subjecthaving cancer, comprising administering to a subject in need oftreatment one or more formulations of tagged proteins, wherein thetagged proteins bind a cancer cell, and administering one or moretherapeutically-effective populations of AT-CAR-expressing T cells,wherein the AT-CAR-expressing T cells bind the tagged proteins andinduce cancer cell death. The term “cancer” is intended to be broadlyinterpreted and it encompasses all aspects of abnormal cell growthand/or cell division. Examples include: carcinoma, including but notlimited to adenocarcinoma, squamous cell carcinoma, adenosquamouscarcinoma, anaplastic carcinoma, large cell carcinoma, small cellcarcinoma, and cancer of the skin, breast, prostate, bladder, vagina,cervix, uterus, liver, kidney, pancreas, spleen, lung, trachea, bronchi,colon, small intestine, stomach, esophagus, gall bladder; sarcoma,including but not limited to chondrosarcoma, Ewing's sarcoma, malignanthemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissuesarcoma, and cancers of bone, cartilage, fat, muscle, vascular, andhematopoietic tissues; lymphoma and leukemia, including but not limitedto mature B cell neoplasms, such as chronic lymphocytic leukemia/smalllymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphomas, andplasma cell neoplasms, mature T cell and natural killer (NK) cellneoplasms, such as T cell prolymphocytic leukemia, T cell large granularlymphocytic leukemia, aggressive NK cell leukemia, and adult T cellleukemia/lymphoma, Hodgkin lymphomas, and immunodeficiency-associatedlymphoproliferative disorders; germ cell tumors, including but notlimited to testicular and ovarian cancer; blastoma, including but notlimited to hepatoblastoma, medulloblastoma, nephroblastoma,neuroblastoma, pancreatoblastoma, leuropulmonary blastoma andretinoblastoma. The term also encompasses benign tumors.

As used herein, the terms “treat”, “treating”, and “treatment” havetheir ordinary and customary meanings, and include one or more of:blocking, ameliorating, or decreasing in severity and/or frequency asymptom of cancer in a subject, and/or inhibiting the growth, division,spread, or proliferation of cancer cells, or progression of cancer(e.g., emergence of new tumors) in a subject. Treatment means blocking,ameliorating, decreasing, or inhibiting by about 1% to about 100% versusa subject in which the methods of the present invention have not beenpracticed. Preferably, the blocking, ameliorating, decreasing, orinhibiting is about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%,50%, 40%, 30%, 20%, 10%, 5% or 1% versus a subject in which the methodsof the present invention have not been practiced.

Administration frequencies of both formulations comprising populationsof AT-CAR-expressing T cells and formulations of tagged proteins willvary depending on factors that include the disease being treated, theelements comprising the AT-CAR-expressing T cells and the taggedproteins, and the modes of administration. Each formulation may beindependently administered 4, 3, 2 or once daily, every other day, everythird day, every fourth day, every fifth day, every sixth day, onceweekly, every eight days, every nine days, every ten days, bi-weekly,monthly and bi-monthly.

The duration of treatment will be based on the disease being treated andwill be best determined by the attending physician. However,continuation of treatment is contemplated to last for a number of days,weeks, or months.

The present invention offers flexibility in the methods of treatment,and as a result, the formulation(s) of tagged proteins and thepopulation(s) of AT-CAR-expressing T cells may be administered to asubject in any order. Thus, the formulation(s) of tagged proteins may beadministered to a subject before, after or concurrently with thepopulation(s) of AT-CAR-expressing T cells. Alternatively, where morethan one formulation of tagged proteins and/or more than one populationof AT-CAR-expressing T cells are administered to a subject, theadministration can be staggered. For example, a first formulation oftagged proteins can be administered, followed by a first population ofAT-CAR-expressing T cells, which is then followed by a secondformulation of tagged proteins and then a second population ofAT-CAR-expressing T cells.

The present invention also includes methods whereby a population ofAT-CAR-expressing T cells is coated with tagged proteins prior toadministration of the AT-CAR-expressing T cells to the subject.

In each of the embodiments of the present invention the subjectreceiving treatment is a human or non-human animal, e.g., a non-humanprimate, bird, horse, cow, goat, sheep, a companion animal, such as adog, cat or rodent, or other mammal.

The invention also provides a kit comprising one or more containersfilled with one or more populations of AT-CAR-expressing T cells and oneor more formulations of tagged proteins. The kit may also includeinstructions for use. Associated with the kit may further be a notice inthe form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, which noticereflects approval by the agency of manufacture, use or sale for humanadministration.

IV. Examples

Mice and cell lines. NOD-scid IL2Rgamma^(null) (NSG) mice were purchasedfrom the Jackson Laboratory (Bar Harbor, Me., USA), housed in theUniversity of Maryland, Baltimore, specific pathogen-free animalfacility and used as recipients for adoptive immunotherapy. Experimentswere reviewed and approved by the University of Maryland BaltimoreInstitutional Animal Care and Use Committee. The human EGFR+ colonadenocarcinoma cell line SW480 (ATCC, Manassas, Va.) was maintained inDulbecco's modified Eagle's medium (DMEM) (GIBCO brand; Invitrogen,Carlsbad, Calif., USA) supplemented with 10% heat inactivated fetalbovine serum (Gemini Bio-Products, West Sacramento, Calif., USA), 2 mML-glutamine (GIBCO brand; Invitrogen) and 1% penicillin-streptomycin(GIBCO brand; Invitrogen). The EGFR+ HER2+ pancreatic adenocarcinomacell line Panc 6.03 was kindly provided by Dr. Elizabeth Jaffee (SidneyKimmel Cancer Center at Johns Hopkins), and cultured in RPMI 1640 medium(GIBCO brand; Invitrogen) supplemented with 20% FBS, 1% MEMnon-essential amino acids (GIBCO brand; Invitrogen), 1% sodium pyruvate(GIBCO brand; Invitrogen), 2 mM L-glutamine, 1% penicillin-streptomycinand 100 IU/ml insulin. The HER-2+ breast adenocarcinoma cell line AU565(ATCC) was cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mML-glutamine and 1% penicillin-streptomycin. The Phoenix Ampho packagingcell line was purchased from Orbigen (San Diego, Calif., USA) andmaintained in D10 medium containing DMEM, 10% FBS, 1% sodium pyruvate, 2mM L-glutamine and 1% penicillin-streptomycin. The surface expression ofEGFR or HER2 on SW480, Panc 6.03 and AU565 cell lines was determined byflow cytometry using FITC-conjugated Cetuximab (Ctx) or FITC-conjugatedHerceptin (Her2).

Construction of retroviral vector. The retroviral vector backbonepMSGV1-CD8-28BBZ (Hughes M. S. et al., Transfer of a TCR gene derivedfrom a patient with a marked antitumor response conveys highly activeT-cell effector functions. Hum Gene Ther 2005 April; 16(4):457-72) was akind gift from Dr. Richard Morgan (National Cancer Institute) and isderived from pMSGV (murine stem cell virus-based splice-gag vector).FIG. 1A shows schematically representation of the vector construct, andthe order of placement of components in-frame from the 5′ to the 3′ends. The mouse scFv against FITC is referred to αFITC-CAR and is linkedto the hinge and transmembrane regions of the human CD8α chain(nucleotide sequence 1271-1519, Genbank NM 001768.6), and thecytoplasmic regions of the human CD28 (nucleotide sequence 760-882,Genbank NM 006139.2), 4-1BB (nucleotide sequence 886-1026, Genbank NM001561.5) and CD3 (nucleotide sequence 299-637, Genbank NM 000734.3)molecules. The αFITC-CAR sequence was synthesized by BlueHeron (Bothell,Wash.).

The sequence was confirmed by DNA sequencing and has the followingsequence:agttgcctgttaggttgttggtgctgatgttctggattcctgatccagcagtgatgtcgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagccttgtacacagtaatggaaacacctatttacgttggtacctgcagaagccaggccagtctccaaaggtcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaaagtagtgctgatgatgctaagaaggatgctgctaagaaggatgatgctaagaaggatgatgctaagaaggatggtgaggtgaagctggatgagactggaggaggcttggtgcaacctgggaggcccatgaaactctcctgtgttgcctctggattcacttttagtgactactggatgaactgggtccgccagtctccagagaaaggactggagtgggtagcacaaattagaaacaaaccttataattatgaaacatattattcagattctgtgaaaggcagattcaccatctcaagagatgattccaaaagtagtgtctacctgcaaatgaacaacttaagagttgaagacatgggtatctattactgtacgggttatactatggtatggactactggggtcaaggaacctcagtcaccgtctcc(SEQ ID NO:1). The sequence was ligated into pMSGV1 to generate theαFITC-CAR retroviral vector.

Retroviral transduction of human T cells. HLA-A2+peripheral bloodmononuclear cells (PBMC) from healthy donors were purchased fromBiological Specialty Corp (Colmar, Pa., USA), and isolated byFicoll-Pague (GE Healthcare, Piscataway, N.J., USA) density gradientcentrifugation. Isolated PBMC were cultured at 3×10⁶ per well in 24-welltissue culture plates in AIM V medium (GIBCO brand; Invitrogen)supplemented with 5% human AB serum (Sigma-Aldrich), 1% MEMnon-essential amino acids, 1% penicillin-streptomycin and 100 U/mlrecombinant human IL-2 (BioLegend, San Diego, Calif., USA), andactivated with 50 ng/ml OKT3 (eBioscience, San Diego, Calif., USA). Twodays later, cells were collected for retroviral transduction. Fortransduction, 24-well non-tissue culture treated plates (BD Biosciences,Franklin Lakes, N.J., USA) were coated with 0.5 ml per well of 10 μg/mlrecombinant human fibronectin fragment (RetroNectin; Takara, Otsu,Shiga, Japan) overnight at 4° C. After incubation, wells were blockedwith 1 ml of Hanks's balanced salt solution (GIBCO brand; Invitrogen)plus 2.5% human AB serum for 30 min at RT, and washed with Hanks'sbalanced salt solution plus 2.5%N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (GIBCObrand; Invitrogen). Transductions were conducted as previously described(Johnson et al. Blood 114, 535-546 (2009)). Briefly, approximately 2.5ml of retroviral supernatant were added to each coated well followed bycentrifugation at 2000 g for 2 h at 32° C. 1.5 ml of viral supernatantwas removed and 1×10⁶ (0.5 ml) activated PBMC were added to each well inthe presence of 100 U/ml IL-2. Plates were centrifugated at 1000 g for10 min, and then incubated overnight at 37° C. After transduction, cellswere washed and maintained in the presence of IL-2 (100 U/ml) and usedin experiments five days after transduction. The surface expression ofαFITC-CAR on transduced human T cells was determined by flow cytometryafter staining cells with CD3 or CD8 and FITC-conjugated cetuximab. Insome experiments, αFITC-CAR transduced T cells were stained withFITC-conjugated dextran beads. Cells stained with FITC-conjugatedpurified human IgG (Invitrogen) were used as an isotype control.

T cell proliferation assay, cytokine and chemokine production assay.Three to five days after transduction, 1×10⁵ T cells were cultured in96-well round-bottom plates coated with cetuximab, FITC-conjugatedcetuximab, or FITC-Dextran for 72 h. For T cell specific reactivityagainst tumor cells, SW480 cells were pulsed with the indicatedconcentrations of antibodies for 1 h at 37° C., washed 3 times. 1×10⁵effector T cells and 1×10⁵ tumor cells were co-cultured in 200 μl ofculture volume in 96-well round-bottom plates for 72 h. Sixteen hoursbefore harvesting, 0.5 μCi of 3H-thymidine was added to each well priorto measuring thymidine uptake using a 1450 LSC & luminescence counter(PerkinElmer, Waltham, Mass., USA). Cytokine and chemokine productionlevels were measured from culture supernatants collected 48 hours afterstimulation using a Cytokine/Chemokine kit (Millipore, Billerica, Mass.,USA) according to manufacturer's instructions. In some experiments,tumor explants were finely minced and cultured in trypsin for 2 hours at37° C. followed by T cell enrichment using a negative selection kit(Invitrogen/Life Technologies, Grand Island, N.Y.). 200,000 T cells wereco-cultured with SW480 (50,000) colon cancer cells which had been pulsedwith FITC-Ctx or Ctx (0.5 μg/1×10⁶ cells). Three days laterproliferation was determined by [³H]thymidine uptake (±SD) or cytokineand chemokine production was measured using a Milliplex array.

Cytotoxicity assay. Cytotoxic activity against tumor target cells wasmeasured using a standard ⁵¹Cr release assay. 1×10⁶ target cells werelabeled with 200 μCi of ⁵¹Cr for 2 h at 37° C., washed 3 times, andpulsed with anti-human antibodies for 1 h at 37° C. 1×10⁴ labeled targetcells were then co-cultured with decreasing numbers of effector T cellsat the indicated effector to target (E:T) ratios in 200 μl of culturevolume in 96-well round-bottom plates. Target cells incubated in mediumalone were used to determined spontaneous ⁵¹Cr release, and maximalrelease was determined by incubating labeled target cells in 10% TritonX-100. After 5 hours at 37° C., 50 μl of supernatant was collected and⁵¹Cr radioactivity was measured in a 1450 LSC & luminescence counter.The mean percentage of specific lysis was calculated according to thefollowing equation: % specific lysis=(test release−spontaneousrelease)/(maximal release−spontaneous release)×100. All tests wereperformed in triplicate wells and results are shown as mean±SD.

Tumor models and adoptive immunotherapy. In prophylactic tumor models 6-to 8-wk-old male NSG mice (n=5 for each group) were injectedsubcutaneously (s.c.) in the rear leg flank with 1−2×10⁶ SW480 or Panc6.03 tumor cells. One day later mice were injected intraperitoneally(i.p.) with FITC-Ctx or Ctx (25 μg/mouse). One day after Ctx injection,mice were injected intravenous (i.v.) with 5×10⁶ αFITC-CAR transducedhuman T cells. After adoptive T cells transfer, mice were injected i.p.with antibodies (25 μg/mouse) weekly for three weeks. Tumor area wasmeasured with digital calipers in a blinded manner two to three timesper week, and tumor sizes (mm²) were calculated by perpendicularmeasurement by longitudinal diameter. Mice were euthanized when tumorsizes reach 200 mm² or if mice became moribund or had troubleambulating. All experiments were performed independently at least twicewith similar results. Survival data were analyzed with the exactlong-rank test.

Experiment 1. Peripheral blood mononuclear cells were activated withanti-CD3 mAb in the presence of IL-2 followed by transduction with theα-FITC-CD28-41BB-CD3ζ-CAR vector (referred to as αFITC-CAR) as shown inFIG. 1A and as described above. The expression of the αFITC scFv on Tcells was analyzed by staining cells with anti-CD8 (or anti-CD3) andFITC-labeled cetuximab (FITC-Ctx) or FITC-labeled dextran (FITC-Dex)beads. On average, 60% of total T cells expressed αFITC-CAR (FIG. 1B).To confirm their functionality and specificity, αFITC-CAR or control(mock transduced) T cells were activated using titrating concentrationsof plate-bound FITC-Ctx, unbound cetuximab, or FITC-Dex beads. αFITC-CART cells proliferated vigorously and in a dose dependent manner followingstimulation with FITC-Ctx and FITC-Dex but did not divide in response tostimulation with Ctx alone (FIG. 1C, left panel). In contrast, control Tcells did not proliferate to FITC-Ctx or FITC-Dex (FIG. 1C, rightpanel). T cells were also co-cultured with EGFR⁺ colon cancer cells(SW480) which had been stained with titrating concentrations ofFITC-Ctx. FITC reactivity by αFITC-CAR T cells was demonstrated by theirability to divide following activation with FITC-Ctx-stained cancercells, FIG. 1D. However, proliferation was similar to that of control atthe lowest concentrations of FITC-Ctx. Control T cells did notproliferative in response to any concentration of FITC-Ctx-stainedcancer cells, FIG. 1D.

To determine their cytolytic capacity, αFITC-CAR T cells were culturedtogether with FITC-Ctx-stained SW480 colon cancer cells at variouseffector to target ratios. αFITC-CAR T cells lysed SW480 colon cancercells at effector to target ratios as low as one T cell to twenty targetcells (FIG. 1E, left panel) but did not lyse cancer cells labeled withFITC-mouse IgG (FIG. 1E, right panel). Similarly, control T cells didnot show an appreciable level of cytolytic activity againstFITC-Ctx-labeled or FITC-IgG-labeled SW480 cancer cells (FIG. 1E). Toconfirm their ability to recognize a variety of target cells expressingdifferent antigens αFITC-CAR T cells were co-cultured with pancreaticcancer cells (Panc 6.03) stained with FITC-Ctx or stained withFITC-Herceptin (anti-Her-2 mAb; FITC-Her2) and breast cancer cells(AU565) stained with FITC-Her2. αFITC-CAR T cells efficiently andspecifically lysed cancer cells stained with FITC-Ctx or FITC-Her2 (FIG.1F). It is worth noting that the increased cytolytic activity againstFITC-Her2-stained pancreatic cancer cells above cells stained withFITC-Ctx is likely associated with the higher expression level Her-2 ascompared with EGFR (data not shown). Furthermore, αFITC-CAR T cellsproduced a wide array of cytokines beneficial to T cell survival,expansion and chemotaxis (Table 1). The background cytokine levelsproduced by control T cells activated with FITC-Ctx- or Ctx-stainedcancer cells were identical to one another.

TABLE 1 Cytokines and chemokine production by αFITC-CAR T cells. HLA-A2⁺PBMCs were activated with anti-CD3 antibodies in the presence of IL-2followed by transduction with αFITC-CAR retrovirus. αFITC-CARfunctionality on transduced was determined by their ability to producecytokines and chemokines following activation with SW480colon cancercells that were stained with FITC-Ctx or Ctx. The levels of variouscytokines and chemokines produced by α-FITC-CAR T cells were measured 72hours after stimulation using a Milliplex cytokine/chemokine array.These data are representative of three independent experiments (threedifferent donors) with each experiment yielding the same trends. Fold+Ctx +FITC-Ctx change FGF-2 1.9 ± (0.79) 15.8 ± (2.59) 8.1 GM-CSF 45.7 ±(5.26)  2803.3 ± (57.74)  61.2 IL-2 13.4 ± (2.6)  193 ± (8.5)  14.4 IL-31.1 ± (0.22) 31.4 ± (6.50) 26.5 IL-5 1.7 ± (0.01)  6.3 ± (0.07) 3.6 IL-70.7 ± (0.64)  2.2 ± (0.32) 3.0 IL-9 0.6 ± (1.06)  1.8 ± (0.25) 2.9 IL-12p70 0.3 ± (0.59)  1.0 ± (0.10) 3.1 IL-13 0.3 ± (0.62) 311.6 ± (6.51) 873.8 IL-17 16.6 ± (2.15)  644.6 ± (20.84) 38.6 sIL-2Ra 61.4 ± (5.18) 919.6 ± (59.03) 14.9 sCD40L 27.6 ± (5.92)  1406.6 ± (158.22) 50.90 IFN-γ4.3 ± (0.98) 2906.6 ± (162.58) 662.1 TNF-α 4.5 ± (0.41) 395.6 ± (33.26)87.8 TNF-β 1.3 ± (0.10) 81.1 ± (7.71) 61.4 MCP-1 (CCL2) 5.1 ± (0.53)2243.3 ± (284.31  437.8 MIP-1α(CCL3) 6.7 ± (0.89) 3234.3 ± (225.02)483.8 MIP-1β(CCL4) 7.8 ± (1.39) 986.3 ± (40.99) 125.3 RANTES (CCL5) 0778.3 ± (66.37) 778 MCP-3 (CCL7) 2.0 ± (1.77) 194.3 ± (4.73)  94.9 MDC(CCL22) 136.3 ± (13.65)  6503.3 ± (551.75) 47.7 Eotaxin (CCL11) 3.2 ±(1.29) 32.1 ± (3.56) 9.8 IP-10 (CXCL10) 14.9 ± (1.60 ) 14166.6 ±(1484.3)  950.7

Collectively, these data demonstrate: 1) the functionality of αFITC-CART cells, 2) their specificity against FITC-Ab-stained cells but notsoluble FITC, 3) their ability to lyse a diverse set tumor cell types,and 4) the use of various FITC-tagged antibodies.

Experiment 2. The ability to re-direct αFITC-CAR T cells to eliminatetumor cells in vivo was examined. Mice were injected s.c. with SW840colon cancer cells followed by the administration of FITC-Ctx or Ctx viai.p. injection. One day later αFITC-CAR T cells were administered viai.v. injection into the tail vein. Tumor growth kinetics was similarbetween mice receiving αFITC-CAR T cells plus Ctx and those receivingCtx alone, FIG. 2A left panel. In sharp contrast, tumor growth wasgreatly suppressed in mice receiving αFITC-CAR T cells plus FITC-Ctx(FIG. 2A left panel). Likewise, the tumor-free occurrence (FIG. 2Amiddle panel) and overall survival (FIG. 2A right panel) wassignificantly improved in mice receiving αFITC-CAR T cells plus FITC-Ctxas compared with control groups. In spite of this survival advantagehowever, mice receiving α-ITC-CAR T cells plus FITC-Ctx succumbed totumor challenge within 55 days of tumor implantation.

The mechanisms contributing to the failure of αFITC-CAR T cells inlong-term treatment were further investigated. Because T cells wereactivated with CD3 mAb and IL-2, CAR T cells can display a shortenedlife despite receiving prosurvival signals from CD28 or 41BB (Sadelainet al. Curr. Opin. Immunol. 21, 215-223 (2009); Brentjens et al. Nat.Med. 9, 279-286 (2003)). The presence of αFITC-CAR T cells in varioustissues was assayed, including the spleen, liver, bone marrow,circulation and in tumor explants. αFITC-CAR T cells were found in alltissues analyzed. Approximately 10% of all human T cells detected intumor explants were αFITC-CAR T cells, FIG. 2B. Similar percentages werefound in the other tissues (data not shown). However, the overallpercentage of αFITC-CAR T cells at the time of organ collection (betweendays 38 and 45) was significantly lower than the starting percentage of60% at infusion. It is also worth noting that 60% to 90% of the T cellsrecovered from mice were CD8⁺, as compared with the starting percentageof 40-50%. These data suggest a potential survival advantage of CD8⁻subset over CD4⁺ T cells in vivo. The frequency or role for CD4 T_(Regs)was not examined but remains a viable option that merits furtherinvestigation.

Alternatively, αFITC-CAR T cells might not have become sufficientlyactivated by antigen (FITC) perhaps due to anergy or other suppressivemechanisms. Total T cells were enriched from the tumor explant andimmediately reactivated using SW480 cells from tissue culture that werestained with FITC-Ctx or Ctx alone. αFITC-CAR T cells proliferated (FIG.2C) and produced various effector molecules and chemokines (FIG. 2D)following stimulation with FITC-Ctx-stained SW480 cells but did notrespond to SW480 cells stained with Ctx. Unfortunately, the numbers ofαFITC-CAR T cells isolated from tumor explants were not sufficient totest their killing aptitude. However, based on their ability to divideand produce effector molecules ex vivo, α-FITC-CAR T cells are clearlycapable of responding to FITC stimulation.

Based on the observations that α-FITC-CAR were present in mice and wereresponsive to FITC-CAR stimulation, EGFR expression on tumor explantswas examined. As examined by flow cytometry, all tumor explants werecompletely devoid of EGFR expression as compared with isotype controland with SW480 cells taken from tissue culture (FIG. 2E). In addition,although the majority of SW480 cells taken from tissue culture expressedEGFR, its expression was heterogeneous with some cells lacking EGFR.

Taken together, these data support the contention that αFITC-CAR killedEGFR⁺ cells but in time allowed the growth of EGFR⁻ cells which were nolonger a target for αFITC-CAR T cells. The lack of EGFR expression, andtherefore lack of FITC-mediated stimulation, might have also contributedto the lower frequency of αFITC-CAR T cells observed at later timepoints in tumor-bearing mice as compared with the percentage ofαFITC-CAR T cells prior to injection. Additionally, these data highlightthe potential for tumor escape occurring in patients in whose TAA isheterogeneously expressed. These studies also emphasize the potentialneed to use CAR T cells with specificity to several TAAs. One advantageto the use of an anti-tag CAR T cells is the potential to use severalFITC ‘tagged’ tumor-reactive antibodies simultaneously. Alternatively,the use of CARs expressing scFvs specific for biotin or PE-conjugatedantibodies would add to the diversity of anti-tag CARs.

Experiment 3. Given the heterogeneity of EGFR expression on SW480 cancercells, the ability of αFITC-CAR T cells to destroy a population ofcancer cells in which all of the cells expressed the antigen wasexamined. The pancreatic cancer cell line Panc 6.03 was selected as thecells uniformly express EGFR (FIG. 3A). The cytolytic activity ofαFITC-CAR T cells was examined in a prophylactic tumor model using thesame procedure described in Experiment 2. Tumor growth was clearlysuppressed in mice receiving αFITC-CAR T cells plus FITC-Ctx whereasmice receiving α-FITC-CAR T cells plus Ctx demonstrated rapid tumorgrowth, FIG. 3B left panel. All mice receiving αFITC-CAR T cells plusCtx succumbed to tumor challenge within 35 days of tumor implantation,FIG. 3B right panel. It is worth noting that the administration ofFITC-Ctx via i.v., i.p., or intratumoral injection all resulted inantibody localization to the tumor (data not shown). An alternativemethod to redirecting T cells to the tumor is to coat αFITC-CAR T cellswith FITC-Ctx prior to adaptive transfer.

Experiment 4. The ability of αFITC-CAR T cells to destroy an establishedpancreatic tumor was examined. Pancreatic tumors were grown to sizesbetween 3-10 mm² when tumors were well-vascularized and then injected(i.p.) with FITC-Ctx or Ctx. One day later mice were administeredαFITC-CAR T cells via tail vein injection. T cells with redirectedspecificity for EGFR eradicated established pancreatic tumors in allmice (FIG. 3C left panel) and improved survival as compared with micetreated with Ctx and αFITC-CAR T cells (FIG. 3C right panel). No tumorrelapse occurred during the time of observation.

In summary, these studies are the first to describe the generation of auniversal and adaptable CAR system which confers T cells specificity toFITC-tagged antibodies which when bound to various cancer types mediatetumor destruction. This report is also the first to emphasize theimportance of using CAR T cells to target more than one TAA asTAA-negative tumor variants can arise and eventually kill the host. Theplatform is considered an ‘off-the-shelf’ system that considerablyadvances the existing CAR technology through its potential to target anassortment of tagged proteins (i.e., antibodies) in order to targetvarious cancer types.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

Throughout this application, various publications, patents, and/orpatent applications are referenced in order to more fully describe thestate of the art to which this invention pertains. The disclosures ofthese publications, patents, and/or patent applications are hereinincorporated by reference in their entireties to the same extent as ifeach independent publication, patent, and/or patent application wasspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A method of treating cancer in a subject,comprising: (a) administering one or more formulations of taggedproteins to a subject in need of treatment, wherein the tagged proteinsbind a cancer cell in the subject, and wherein the tagged proteins areantibodies or antigen-binding fragments thereof, and (b) administeringone or more therapeutically-effective populations of anti-tag chimericreceptor (AT-CAR)-expressing T cells to the subject, wherein the AT-CARcomprises a tag-binding domain, a transmembrane domain, and a T cellactivation domain, where the T cell activation domain consists of one ormore of the cytoplasmic region of OX40, the cytoplasmic region of HVEM,and FcRε, where the T cell activation domain does not comprise CD3ζ, andwherein the AT-CAR-expressing T cells bind the tagged proteins andinduce cancer cell death, thereby treating cancer in a subject.
 2. Amethod of treating cancer in a subject, comprising: (a) administering atleast two formulations of tagged proteins to a subject in need oftreatment, wherein the tagged proteins bind a cancer cell in thesubject, and wherein the tagged proteins are antibodies orantigen-binding fragments thereof, and (b) administering at least twotherapeutically-effective populations of AT-CAR-expressing T cells tothe subject, wherein the AT-CAR comprises a tag-binding domain, atransmembrane domain, and a T cell activation domain, where the T cellactivation domain consists of one or more of the cytoplasmic region ofOX40, the cytoplasmic region of HVEM, and FcRε, where the T cellactivation domain does not comprise CD3ζ and wherein theAT-CAR-expressing T cells bind the tagged proteins and induce cancercell death, thereby treating cancer in a subject.
 3. The method of claim1, wherein the tags of the formulations of tagged proteins are the sameor different and the tags are selected from the group consisting offluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol,peridinin chlorophyll protein complex, green fluorescent protein,phycoerythrin (PE), horse radish peroxidase, palmitoylation,nitrosylation, alkalanine phosphatase, glucose oxidase, and maltosebinding protein.
 4. The method of claim 1, wherein the antibodies orantigen-binding fragments thereof of the formulations are the same ordifferent.
 5. The method of claim 4, wherein the antibodies orantigen-binding fragments thereof are selected from the group consistingof cetuximab, nimotuzumab, panitumumab, retuximab, omalizumab,tositumomab, trastuzumab, gemtuzumab, alemtuzumab or an antigen-bindingfragment of any one thereof.
 6. The method of claim 1, wherein theAT-CAR of the AT-CAR-expressing T cells are the same or different. 7.The method of claim 6, wherein the tag-binding domain is an antibody oran antigen-binding fragment thereof.
 8. The method of claim 6, whereinthe tag-binding domain specifically binds FITC, biotin, PE orstreptavidin.
 9. The method of claim 7, wherein the antigen-bindingfragment is a single chain variable fragment (scFv).
 10. The method ofclaim 7, wherein the antigen-binding fragment is a single chain variablefragment (scFv) that specifically binds FITC, biotin, PE orstreptavidin.
 11. The method of claim 6, wherein the transmembranedomain is the hinge and transmembrane regions of the human CD8a chain.12. The method of claim 1, wherein the T cells of the populations ofAT-CAR-expressing T cells are the same or different and wherein the Tcells are selected from the group consisting of T cells of anyHLA-background from peripheral blood mononuclear cells (PBMC), T cellsisolated from a tumor explant of the subject, and intratumoral T cellsof the subject.
 13. The method of claim 1, wherein the T cells of thepopulations of AT-CAR-expressing T cells consist of HLA-A2+peripheralblood mononuclear cells (PBMC).
 14. The method of claim 1, wherein theformulations of tagged proteins are administered to the subject prior toor after administration of the therapeutically-effective populations ofAT-CAR-expressing T cells.
 15. The method of claim 1, wherein theformulations of tagged proteins are administered to the subjectconcurrently with administration of the therapeutically-effectivepopulations of AT-CAR-expressing T cells.
 16. The method of claim 1,wherein the formulations of tagged proteins and thetherapeutically-effective populations of AT-CAR-expressing T cells areadministered to the subject in any order.
 17. The method of claim 1,wherein AT-CAR-expressing T cell binding to the tagged proteins inducescytolytic activation of the T cells.
 18. The method of claim 1, whereinthe subject is a human.